Personalized medicine promises to revolutionize clinical care by providing more accurate diagnoses, better targeted therapies, and response monitoring. Approaches to personalized medicine can benefit from determining which particular disease configuration a patient has through the application of one or more stains to a tissue sample taken from the patient, and subsequent assessment of the stained tissue sample.
Biological stains are generally used in tissue histology and other fields to enhance contrast between particular structures of interest in tissues and the remainder of a tissue section. Such contrast is typically observed when stained tissue samples are viewed under an optical microscope. Certain immunohistochemical (IHC) stains bind selectively to certain structures and/or cell types within a tissue sample. Under a microscope, the color of regions that include stain molecules bound to tissue structures can be different from the color of unstained regions of the sample. The resulting color contrast can allow a trained operator to make a visual assessment of the sample.
Anatomical and surgical pathology rely heavily on visual assessment of stained clinical tissue sections. Commonly used stains such as hematoxylin and eosin (H&E) achieve specificity according to the manner in which the stain interacts with components of tissue sections. For example, stains can be applied to selectively stain disease-related proteins and other components in tissue sections. These stains reveal disease-specific architectures and morphologies which provide cues for visual diagnosis.
Fibrosis results from wound-healing responses to chronic tissue injury in a variety of tissues. For example, renal fibrosis is a progressive process that can lead to end-stage renal failure, which may require dialysis and/or kidney transplantation. Liver fibrosis can result in cirrhosis, portal hypertension, and liver failure, and may require liver transplantation. The assessment of the severity of old and/or active fibrotic processes in subjects is important in diagnosing a variety of fibrosis-related conditions, and in evaluating subject responses to antifibrotic therapy.
Fibrosis is characterized by excessive accumulation of extracellular matrix constituents including collagens, proteoglycans, and glycoproteins. One method for assessment of fibrosis activity is by examining tissue sections (e.g., liver biopsy sections) for the presence of collagen under an optical microscope. However, common general purpose histological stains such as H&E do not provide reliable quantitative information about collagen accumulation under typical assessment conditions. Instead, special stains such as Masson's trichrome stain and picosirius red are typically used to measure fibrosis stage in tissue sections.
Picosirius red is birefringent and collagen-specific, and chemically enhances the birefringence of collagen fibers when it binds to the fibers. Bound picosirius red can increase the birefringence of collagen fibers so that relative amounts or types of collagen can be discerned as colors under a conventional white light polarized light microscope with crossed circular polarizers and broadband illumination (e.g., several hundreds of nanometers). For example, larger collagen fibers may appear bright yellow or orange, while smaller fibers may appear green.
Examination of tissue sections typically begins with a pathologist performing a visual assessment of an H&E-stained sample, and deciding that further assessment with a special stain is needed. Another sample is then stained with trichrome (which demarks collagen with a blue color to aid in visual assessment), or occasionally with picosirius red, and viewed under a polarized light microscope equipped with crossed circular polarizers. One of the polarizers can be slightly detuned so that the microscope's background image field is not completely dark. Images of the second sample are recorded, and collagen identified in the second sample can be qualitatively categorized based on visual inspection of its color in the images.